Structural and functional characterization of Mpp75Aa1.1, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm

Abstract

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (β-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.

Safety assessment of insecticidal proteins: A case study utilizing His-tagged protein to determine the safety of Mpp75Aa1.1, a new ETX_MTX2 protein that controls western corn rootworm

“…The presence of a His tag had no impact on the mammalian safety of Mpp75Aa1.1 suggesting that the use of His-tagged proteins is appropriate for use in mouse acute toxicity studies. Thus, the toxicological study results are consistent with the conclusions of safety based upon functional domain-based characterization (18, 19).…”

“…The EG4227 strain was isolated from grain dust while the BOD strain was isolated from the powder of a commercially available probiotic capsule. Cell cultures for each strain and DNA extractions methods were previously reported (11, 18). The Mpp75A genes were cloned from the EG4227 and BOD strain, respectively and the two amino acid sequences, deduced from the gene sequence, were designated as Mpp75Ab1 and Mpp75Ab2 by the BPPRC nomenclature committee using methods previously described (11).…”

“…Primary amino acid sequence and structural homology have identified Mpp75Aa1.1 as belonging to the Clostridium epsilon toxin ETX/ Bacillus mosquitocidal toxin MTX2 (ETX_MTX2) protein family (Pfam:PF03318) (17, 18). The ETX_MTX2 protein family is one of β-pore forming protein (β−PFPs) families, characterized by a shared common mode of action (MOA) that utilizes an amphipathic hairpin loop to insert into cell membranes to form a β-barrel membrane-integrated pore.…”

“…The results from the ETX_MTX2 protein Mpp51Aa2.834_16 confirmed that the functional specificity and selectivity against targeted insect pests within this protein family is conferred by specific residues within the receptor-binding domain (24, 26). Screening analysis has also suggested that the receptor-binding region of the Mpp75Aa1.1 protein, which is similarly diversified from other ETX_MTX2 proteins, is responsible for its insecticidal specificity (18).…”

Safety assessment of Mpp75Aa1.1, a new ETX_MTX2 protein from Brevibacillus laterosporus that controls western corn rootworm

“…Primary amino acid sequence and structural homology analysis have identified Mpp75Aa1.1 as belonging to the Clostridium epsilon toxin ETX/ Bacillus mosquitocidal toxin MTX2 (ETX_MTX2) protein family (Pfam:PF03318) [ 17 , 18 ]. The ETX_MTX2 protein family is one of the β-pore forming protein (β-PFP) families, characterized by a shared common mode of action (MOA) that utilizes an amphipathic hairpin loop to insert into cell membranes to form a β-barrel membrane-integrated pore.…”